Lanphere, C;
Offenbartl-Stiegert, D;
Dorey, A;
Pugh, G;
Georgiou, E;
Xing, Y;
Burns, JR;
(2021)
Design, assembly, and characterization of membrane-spanning DNA nanopores.
Nature Protocols
, 16
pp. 86-130.
10.1038/s41596-020-0331-7.
Preview |
Text
Howorka_NP for RPS deposit.pdf - Accepted Version Download (854kB) | Preview |
Abstract
DNA nanopores are bio-inspired nanostructures that control molecular transport across lipid bilayer membranes. Researchers can readily engineer the structure and function of DNA nanopores to synergistically combine the strengths of DNA nanotechnology and nanopores. The pores can be harnessed in a wide range of areas, including biosensing, single-molecule chemistry, and single-molecule biophysics, as well as in cell biology and synthetic biology. Here, we provide a protocol for the rational design of nanobarrel-like DNA pores and larger DNA origami nanopores for targeted applications. We discuss strategies for the pores’ chemical modification with lipid anchors to enable them to be inserted into membranes such as small unilamellar vesicles (SUVs) and planar lipid bilayers. The procedure covers the self-assembly of DNA nanopores via thermal annealing, their characterization using gel electrophoresis, purification, and direct visualization with transmission electron microscopy and atomic force microscopy. We also describe a gel assay to determine pore–membrane binding and discuss how to use single-channel current recordings and dye flux assays to confirm transport through the pores. We expect this protocol to take approximately 1 week to complete for DNA nanobarrel pores and 2–3 weeks for DNA origami pores.
| Type: | Article |
|---|---|
| Title: | Design, assembly, and characterization of membrane-spanning DNA nanopores |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1038/s41596-020-0331-7 |
| Publisher version: | https://doi.org/10.1038/s41596-020-0331-7 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10121539 |
Archive Staff Only
 |
View Item |
